Project Summary/Abstract Translation is a crucial point of regulation of eukaryotic gene expression, and dysregulation of translation is implicated in many human diseases. Although it is well known that mRNA levels fail to predict protein levels for most genes, and that mRNA-specific translational efficiencies vary by orders of magnitude, the molecular mechanisms responsible for such widespread and quantitatively significant translational effects on gene expression are poorly understood. Most studies of translational control emphasize functional differences between mRNA features and mRNA-binding factors (e.g. proteins and miRNAs), and consider 'the ribosome' as an unvarying component in the process. This view overlooks provocative evidence suggesting that molecular specialization of ribosome function may also play an important role in regulating gene expression. Translational control through ribosome specialization was first proposed more than thirty years ago. Despite increasing proteomic evidence for the production of growth condition and developmental stage specific ribosomes, the functional consequences of such ribosome 'specializations' have never been rigorously tested. Our lab studies translational regulation in haploid S. cerevisiae undergoing filamentous differentiation, a developmental program induced by prolonged glucose starvation, which serves as a well-established model for environmentally regulated cellular differentiation. Our Preliminary Results demonstrate the suitability of this model system for investigating the biochemical and physiological effects of ribosome specialization on regulation of gene expression. We have obtained preliminary data showing that glucose starved yeast cells produce ribosomal complexes with different protein compositions; that the glucose starved ribosomes have different functional properties; and that starvation-induced changes in ribosome protein composition lead to gene-specific effects on translational efficiency. The proposed work will investigate the mechanisms underlying these interesting effects. Our approach exploits sensitive genome-wide translational profiling methods to determine the scope of translational control effected by specific alterations of ribosomes. By starting with a global perspective, we identify the most physiologically relevant mRNA substrates. We then use these mRNA substrates for in vitro biochemical assays to dissect the molecular mechanisms underlying the global translational responses. This powerful approach, which combines breadth in vivo with mechanistic depth in vitro, is currently underutilized in the translation field. The high degree of conservation of eukaryotic translation mechanisms and regulatory processes argues that the molecular insights we gain from yeast will provide paradigms for understanding translational control in human development and disease. PUBLIC HEALTH RELEVANCE: Project Narrative Translational regulation is essential for human health and development, but only a handful of translational regulatory mechanisms are understood. The proposed work will provide the first detailed understanding of the biochemical and physiological functions of ribosome specialization, an under-studied topic in the translational control field. We anticipate that our results will have broad implications for the study of eukaryotic gene expression, and will also illuminate the etiology of disease states, including cancer, that are associated with dysregulation of ribosome function.